Marine Actinomycetes with Probiotic Potential and Bioactivity against Multidrug-resistant Bacteria

Considering antimicrobial resistance problem, marine microorganisms with the bioactivity against multi-drug resistant (MDR) pathogens have attracted many scientific interests. To address this issue, a total of 21 marine actinomycetes isolated from the Caspian Sea have been screened out. Primary screening via cross-streak method revealed that 3 strains: MN2, MN39, and MN40 produce antimicrobial agents with wide spectrum activity. In the second step, the potent strains were characterized morphologically, and then identified genetically using 16S rRNA analysis. After that, the bioactivity of the ethyl acetate extracts of liquid culture against some MDR bacteria has been studied using disc diffusion method. Finally, the exoenzymatic activity of the strains, and the anti-vibrio activity of the extracts have been evaluated. The nucleotide sequence of the 16S rRNA gene (1.5 kb) showed that the potent strains belong to the genus Streptomyces. The results of disk diffusion method indicated that among the 3 potent isolates, MN39 and MN2 produce biomolecules with antibacterial activity against MDR bacteria specially methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). In addition, potent strains showed remarkable anti-vibrio activity as well as extracellular enzyme production including amylase and protease. The results of this study revealed that the marine actinomycetes isolated from the sediments of Caspian Sea produce biomolecules effective against MDR bacteria, and suggested that these strains deserve to be studied as potential probiotics due to their anti-vibrio activity besides exoenzyme production.

bacteria which are recognized as important secondary metabolite producers (6). Among the Actinobacteria, genus Streptomyces is a powerful producer of functional metabolites with broad range of pharmaceutical activities such as antimicrobial, antitumor, antiviral, and probiotic activity (4,7). In addition, it is apparent that the marine environment could be a source for finding new Actinobacteria with novel bioactive metabolites or biologically active compounds that may be used as biocontrol agents (8,9). Today, there are some studies that have considered marine actinobacteria as potential probiotic in aquaculture since these marine strains are able to inhibit the growth of Vibrio spp. (8,10).
The present study began by screening marine sediments of Caspian Sea for isolating Actinobacteria with antimicrobial activity against MDR bacteria, and then the ability of selected strains to produce exoenzymes as well as their antivibrio activity has been investigated.

Microorganisms
The 21 actinomycete strains used in this work had been isolated before from Caspian Sea sediment (11) and deposited to the microbial laboratory of school of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.

Viability of all strains was maintained on Starch
Casein Agar (SCA) slants at 4 °C. For longer storage, each strain was grown on nutrient broth at 28 °C. After 7 days, glycerol was added to the final concentration of 15% and stored at -20 °C (11).

Indicator microorganisms
The

Sub-merged fermentation
A single colony of selected actinomycetes was inoculated into 50 ml of seed medium (modified

Secondary screening of antimicrobial activity using disk diffusion method
The antimicrobial activities of the concentrated extracts were tested against different indicator microorganisms using agar disc diffusion method as described by Kirby-Bauer with some modifications (5 Table 2 summarizes the spore morphology formed by

Molecular phylogeny of the selected strains
The partial 16S rDNA sequences of the producer strains were determined, and a phylogenetic tree was constructed (Figure 3).
Comparison of ribosomal DNA gene sequence

Sub-merged fermentation
The anti-MDR activities of the crude extracts obtained from the liquid culture of 3 selected isolates were evaluated using disc diffusion method ( Figure 4).
The obtained-crude extracts inhibited the growth of MRSA and VRE more than other indicators which results in forming widest inhibition zone around the impregnated discs (Table3). Table 3. Antibacterial activity of the potential actinomycete isolates using Kirby-Bauer disk diffusion method.

Exoenzyme and anti-vibrio activity
Antagonistic activity of potent isolates has been evaluated against three species of Vibrio.
Although bioactive metabolites produced by these three actinomycetes inhibited the growth of V.
harveyi and V. proteolyticus, but MN2 isolate could not produce any biomolecule which could prevent the growing of V. parahaemolyticus (Table 4). In addition, all three actinomycetes produced extracellular enzymes including amylase and protease. However, the production of amylase by MN39 seemed to be lower.

Discussion
Currently, one of the main threats to the healthcare in the world is the antimicrobial resistance, as stated by WHO (19). To combat this problem, there is an urgent need for probing new drugs with activity against antibiotic-resistant microorganisms. Natural compounds isolated from marine sources may play an important role to lead to the discovery of new antimicrobial agents. Due to the special condition of marine environment, actinomycetes are particularly desirable due to their high ability of producing bioactive compounds (8,9). Additionaly, this solvent is not miscible with water, which results in easy separation of organic from water phase (11,12).
Being a probiotic in an aquaculture may arise from the capability of having antagonistic activity against aqua-pathogens such as Vibrio species (8) or producing extracellular enzymes which help in digesting the nutrients, leading to a better growth of host (9,25). You et al. characterized some marine streptomycetes with anti-vibrio activity, and suggested their potential as probiotics (26). In 2013, the biomass production of 10 Streptomyces with starch-hydrolyzing activity that can be used as probiotics has been studied by Srisamai et al. (27).
In the present study, the anti-vibrio activity of selected actinomycetes besides their ability to produce amylase and protease enzymes have been demonstrated. These findings show the potential of these strains to be adopted as valuable biocontrols, and aiding-growth agents in aquaculture industries.
Although, it should be emphasized that for Streptomyces to be considered as a probiotic and/ or a growth-stimulator factor in aquaculture ecosystems, performing further extensive studies are necessary. marine actinobacteria against multidrug resistance